The goals of this investigation are to determine the genetic mechanisms regulating the expression of the phosphorylase kinase (PhK) subunits (alpha, beta, gamma and delta) and the molecular basis of the I/Lyn mutation in terms of these genetic mechanisms. The following specific aims delineate a practical approach to meet these goals and will require the use of the specific recombinant DNA probes developed. 1. To determine whether there are particular messenger RNA species for any of the PhK subunits that are preferentially expressed in either liver, cadiac, or skeletal muscle tissues. This analysis will involve the hybridization of subunit specific cDNA probes to RNA isolated from the tissues and fractionated on agarose gels. The analysis will also include S1 nuclease digestion of cDNA-RNA hybrids to determine localized regions of heterogeneity in the messages in the different tissues. 2. Analysis of the genomic complexity of the genes encoding each of the PhK subunits. This will involve the hybridization of subunit specific cDNA probes to restriction enzyme digests of genomic DNA fractionated on agarose gels. The results will reveal whether any of the PhK subunits are encoded by multiple genes and will define a partial restriction enzyme map for each of these genes within the mouse genome. 3. Comparison of PhK expression in I/Lyn tisues to that in normal tissues. The analysis described above will also be done on RNA and genomic DNA from I/Lyn tissues. The comparison of the RNA analysis will determine whether there is a particular message for any of the PhK subunits which is altered or absent in the PhK deficient tissue. The genomic analysis may reveal a deletion, insertion, or inversion in one of the PhK subunits' genes which may account for the I/Lyn phenotype. 4. To determine whether the genes for the gamma or alpha subunits are located on the x-chromosome. For this experiment, two human cell lines have been obtained from the Human Genetic Cell Repository. One of these cell lines is haploid for the x-chromosome and the other is tetraploid. By comparing the alpha and delta gene copy number in these two cell lines it can be determined whether either of these genes are on the x-chromosome. The results of this research will advance our understanding of the molecular mechanisms which coordinate expression of multisubunit enzymes and define on the molecular level an inherited metabolic disorder.